Research ArticleScreening and Functional Analysis of Hub MicroRNAs Related to Tumor Development in Colon Cancer

Dong-Hu Yu,1,2 Wei Li,3 Jing-Yu Huang,4,5 Xiao-Ping Liu,2 Chi Zhang,6 Xiao-Lan Ruan ,7 and Sheng Li 1

1 Department of Biological Repositories, Human Genetics Resource Preservation Center of Hubei Province, Zhongnan Hospital of Wuhan University, Wuhan 430071, China

2�e Second Clinical College, Wuhan University, Wuhan 430071, China3Department of Oncology, �e First People’s Hospital of Tianmen, Tianmen 431700, Hubei Province, China4Department of �oracic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China5Hubei Key Laboratory of Tumor Biological Behaviors & Hubei Cancer Clinical Study Center, Wuhan 430071, China6 Department of Gastrointestinal Surgery, Hubei Cancer Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430071, China

7Department of Hematology, Renmin Hospital of Wuhan University, Wuhan 430071, China

Correspondence should be addressed to Xiao-Lan Ruan; 344901800@qq.com and Sheng Li; lisheng-znyy@whu.edu.cn

Dong-Hu Yu and Wei Li contributed equally to this work.

Received 17 June 2019; Accepted 8 August 2019; Published 23 January 2020

Academic Editor: Ernesto S. Nakayasu

Copyright © 2020 Dong-Hu Yu et al. �is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Various microRNAs (miRNAs) are of importance in the development of colon cancer, but most of the mechanisms of the miRNAs are still unclear. In order to clarify the hub miRNAs and their roles in colon cancer development, GSE98406 was used to screen hub miRNAs by bioinformatics analysis. 46 DE-miRNAs (14 were upregulated and 32 were downregulated) and 1738 target genes of DE-miRNAs were ascertained. miRNAs-gene-networks and miRNAs-GO-networks were built to get more knowledge about the function of candidate miRNAs. A�er validation, three miRNAs (miR-17-5p, miR-182-5p and miR-200a-3p) were recognized to be hub miRNAs associated with the progression of colon cancer. More importantly, the hub miRNAs and the putative targets genes might be new diagnostic and therapeutic targets for colon cancer in the future.

1. Introduction

Colon cancer is a common malignancy that affects more than 130,000 people each year, causing about 60,000 deaths [1, 2]. Although the overall five-year survival of patients with colon cancer is generally high with proper treatment, the complex unknown pathogenesis limits the further improvement in colon cancer treatment [3, 4]. �erefore, there is an urgent need for more insights into the pathogenesis of colon cancer. In recent years, miRNAs’ role in cancer research has received increasing attention. miRNA is an important factor in tumor-igenesis and metastasis, and its expression characteristics are closely related to the occurrence, progression, and prognosis of various tumors [5, 6]. Previous studies have identified some important miRNAs impairing the development of cancers by miRNA expression profiles [7–9]. Besides, bioinformatics

analysis was widely used for the identification of novel bio-markers and mechanism studies [10, 11]. In this study, we aimed to search and confirm hub miRNAs that play important parts in the development of colon cancer, thus providing more information for the mechanism research and clinical applica-tion of colon cancer.

2. Materials and Methods

2.1. Data Collection and Processing. �e brief workflow of this study is shown in Figure 1. �e microRNA expression profiles of GSE98406, GSE83924, GSE48267, and GSE35834 were downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). Quantile normalization was performed to normalize all datasets. Table

HindawiBioMed Research InternationalVolume 2020, Article ID 3981931, 8 pageshttps://doi.org/10.1155/2020/3981931

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S1 lists the details of these datasets. GSE98406 was used as a training dataset for screening DE-microRNAs. GSE83924 and GSE48267 were used as independent sample T test sets for verification, respectively. In addition, the clinicopathological correlation analysis for the colon cancer samples in GSE35834 was performed.

2.2. Screening of DE-miRNAs in Colon Cancer Tissues. In this study, the “limma” package in R [12] was used to screen DE-miRNAs between normal colon tissues and tumor tissues. �e cutoff criteria were FDR < 0.05 and |Log2FC| > 1.5.

2.3. Functional Enrichment Analysis of Putative Target Genes. In order to get more knowledge about the candidate miRNAs function, we submitted the selected miRNAs to GCBI to screen their target genes. GCBI is an online tool, which can be used to predict miRNA target genes based on miRanda and TargetScan. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for target genes were performed. �e cut-off criterion is FDR < 0.05. Also, we drew an interactive network by pathway network (path-net) analysis in GCBI, which covered the significant KEGG pathway to find the hub

pathways. �e degrees of each pathway in this path-net were calculated, and the top 5 pathways with highest degrees were selected as hub pathways.

2.4. Identification and Validation of HUB miRNAs. A�er understanding the target genes and the GOs for DE-miRNAs, two important networks for this study (miRNA-gene-network and miRNA-GO-network) were built. Based on the ordering of the number of microRNAs in the two networks, we selected the overlapping hub miRNAs and the key regulatory functions of these miRNA. Two datasets (GSE83924 and GSE48267) were used to verify differential expression levels of these miRNAs between normal colon tissues and tumor tissues by independent sample T test, respectively. �푃 < 0.05 was considered statistically significant. Meanwhile, the datasets of GSE83924 and GSE48267 were used to perform ROC curve analysis, and the AUC for each hub miRNA was calculated to distinguish the tumor tissues from the normal tissues.

2.5. �e Clinical Significance of HUB miRNAs in Colon Cancer. Based on 52 cases of colon cancer samples with complete clinical information in GSE35834, the relationship between the expression of hub miRNAs and clinicopathological

Downloading normalized data ofGSE98406

Data processing

Identi�cation of di�erentially expressed miRNAs(DE-miRNAs) in colon cancer tissues

Identi�cation of putative target genes

Functional and pathway enrichment analysisfor the target genes

miRNAs-gene-networks miRNAs-GO-networks

Hub miRNA identi�cation and validation

Figure 1: Flow chart of data preparation, processing, analysis, and validation.

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parameters was evaluated by clinicopathological correlation analysis. According to the amount of hub miRNA expression, 52 cases were classified into high expression group and low expression group according to the hub miRNA expression (high group, �푛 = 26; low group, �푛 = 26). Chi-square tests were used to assess the relationship between the expression of hub miRNA and gender, age, tumor grade, TNM stage, and metastasis of colon cancer patients. A value of � <0.05 was considered as statistically significant.

3. Results

3.1. DE-miRNAs and Target Genes in Colon Tumor Tissues. Under the thresholds of FDR < 0.05 and |Log2FC| > 1.5, a total of 46 DE-miRNAs (14 up-regulated and 32 down-regulated in colon cancer samples) were selected from 7 control samples and 14 colon samples in GSE98406. �e volcano plot for DE-miRNAs was taken (Figure 2) and the characteristics of the dysregulated miRNAs are listed in Table  1. Based on miRanda and TargetScan, 1738 putative miRNA target genes were identified using GCBI (Table S2).

3.2. Functional Enrichment Analysis of Target Genes. To study the roles of DE-miRNAs in mediating colon cancer progression, we performed GO analysis and KEGG pathway enrichment analysis for target genes. �e data in Table 2 indicate that top 10 GOs were “transcription, DNA-dependent’’, ‘‘regulation of transcription, DNA-dependent’’, ‘‘signal transduction’’, ‘‘positive regulation of transcription from RNA polymerase II promoter’’, ‘‘apoptotic process’’, ‘‘positive regulation of transcription, DNA-dependent’’, ‘‘negative regulation of transcription from RNA polymerase II promoter’’, ‘‘nervous system development’’, ‘‘axon guidance’’ and ‘‘protein phosphorylation’’. According to the KEGG database, the main pathways involving the target genes were demonstrated. As shown in Table 3, the top 10 pathways were “MAPK signaling pathway”, “pathways in cancer”, “PI3K-Akt signaling pathway, proteoglycans in cancer”, “HTLV-I infection”, “endocytosis”, “transcriptional mis-regulation in cancer”, “neurotrophin signaling pathway”,

“axon guidance and GnRH signaling pathway”. What is more, a pathway network was shown, which covers 25 significantly changed pathways (Figure 3). Moreover, the MAPK signaling pathway (degrees:41), apoptosis (degrees:27), pathways in cancer (degree:27), cell cycle (degrees:23) and p53 signaling pathway (degrees:23) showed highest connectivity degrees in path-net, which demonstrated these 5 pathways would play a central role in tumor development.

3.3. Hub miRNAs Identification and Validation. To identify hub miRNAs and their main functions in the development

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Figure 2: �e volcano plot for DE-miRNAs.

Figure 3:  Pathway network (Path-net). Significantly changed pathways were connected in a Path-net to show the interaction network among these pathways. Each pathway in the network was measured by counting the upstream and downstream pathways. �e blue circle represents pathways involving upregulated miRNAs, while the yellow circle represents pathways involving both upregulated and downregulated miRNAs. �e size of the circle represents the degree value and the lines show the interaction between pathways. A higher degree of pathway indicates that it plays a more important role in the signaling network.

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to be hub miRNAs (Table 4). �e bioinformatic analysis showed the hub miRNAs were lowly expressed in colon cancer tissues compared with normal colon tissues. And dysregulated miRNAs play important roles in signal transduction, apoptotic process, and pathways in cancer. GSE83924 and GSE48267 were used to make validation. Further proved by the datasets of GSE83924 and GSE48267, miR-17-5p, miR-182-5p and miR-200a-3p were lowly expressed in tumor tissues (Figure 6). Besides, ROC curve for GSE83924 indicated that miR-17-5p (AUC = 0.918), miR-182-5p (AUC = 0.853) and miR-200a-3p (AUC = 0.783) exhibited excellent diagnostic efficiency for tumor and normal tissues. And ROC curve for GSE48267 also demonstrated that miR-17-5p (AUC = 0.829), miR-182-5p (AUC = 0.849) and miR-200a-3p (AUC = 0.709) exhibited diagnostic efficiency for tumor and normal tissues (Figure 7).

3.4. Association of Hub miRNAs Expression with Clinical Significance. Chi-square analysis for GSE35834 showed that miR-17-5p expression was associated with tumor grade significantly (�푃 = 0.02), and miR-182-5p expression group was associated with advanced TNM stage (III/IV) (�푃 = 0.019). No other significant difference was observed in other clinicopathological features (age, gender, and metastasis) (Table S3).

4. Discussion

In this study, bioinformatics analysis of GSE98406 revealed 46 DE-miRNAs (down-regulated 32 and up-regulated 14). According to the miRNAs-gene-networks and miRNAs-GO-networks, miR-17-5p, miR-182-5p, and miR-200a-3p were considered to be hub miRNAs. �ey play an important role in tumor development as tumor suppressor genes and oncogenes. Although miR-182-5p and miR-200a-3p have

of colon cancer, we selected target genes and DE-miRNAs to construct miRNAs-gene-networks (Figure 4) and miRNAs-GO-networks (Figure 5) according to the significant regulation of GOs and pathways. According to the rank of degrees of miRNAs in two networks, the top rated three miRNAs (miR-17-5p, miR-182-5p, and miR-200a-3p) were determined

Table 1: �e characteristics of the dysregulated miRNAs.

Rank miRNAs FC FDR Feature1 miR-486-5p 5.903792 0.002243 up2 miR-451a 3.594096 0.003163 up3 miR-378i 2.871432 0.006424 up4 miR-126-3p 2.390553 0.007611 up5 miR-378a-3p 2.628745 0.008606 up6 miR-378c 2.505525 0.009621 up7 miR-30a-5p 3.096433 0.011253 up8 miR-378f 2.70463 0.012609 up9 miR-422a 2.655992 0.013226 up10 miR-139-5p 2.804786 0.013957 up11 miR-1280 1.776806 0.034109 up12 miR-4286 1.997162 0.034955 up13 miR-193b-3p 2.347341 0.041491 up14 miR-125a-5p 1.83864 0.042671 up15 miR-182-5p −6.34791 0.003257 down16 miR-3687 −4.03913 0.004112 down17 miR-503-5p −2.85125 0.004506 down18 miR-18b-5p −3.16775 0.005848 down19 miR-4417 −4.53068 0.006188 down20 miR-1246 −4.10737 0.00864 down21 miR-224-5p −2.39326 0.008975 down22 miR-200c-3p −3.51686 0.009565 down23 miR-552-3p −2.65361 0.010763 down24 miR-877-5p −2.15946 0.011788 down25 miR-501-5p −1.64608 0.012554 down26 miR-203a −3.40787 0.013864 down27 miR-146a-5p −2.79816 0.015957 down28 miR-18a-5p −2.98152 0.017964 down29 miR-210-3p −2.21905 0.018626 down30 miR-424-3p −2.14658 0.024091 down31 miR-1290 −2.70448 0.024521 down32 miR-25-5p −2.3023 0.025739 down33 miR-4449 −1.98777 0.026635 down34 miR-3651 −2.38984 0.026706 down35 miR-141-3p −2.92073 0.027618 down36 miR-17-5p −1.97557 0.032908 down37 miR-200a-3p −2.79035 0.037529 down38 miR-188-5p −1.81097 0.04154 down39 miR-106b-3p −2.06644 0.042212 down40 miR-130b-3p −1.84017 0.043567 down41 miR-21-5p −2.79495 0.045308 down42 miR-19a-3p −2.02304 0.045882 down43 miR-3648 −2.51032 0.045897 down44 miR-708-5p −2.86582 0.046121 down45 miR-155-5p −2.09222 0.048755 down46 miR-3175 −2.52047 0.04994 down

Figure 4:  miRNAs-gene-network. According to the interactions between miRNAs and the intersected target genes, miRNAs-gene-network was constructed. �e blue circles represent genes, while blue square nodes represent downregulated miRNAs. �e size of the circle or square node represents the degree value. A higher degree of gene/miRNAs indicates that it plays a more important role in the signaling network.

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(negative regulation of transcription from RNA polymerase II promoter and Positive regulation of transcription from RNA polymerase II promoter). Taken together, the hub miRNAs (hsa-miR-17-5p, hsa-miR-182-5p and hsa-miR-200a-3p) that we identified were reliable, which may be candidate biomarkers for colon cancer.

As for the 3 hub miRNAs, we conducted a literature review of these miRNAs. miR-17-5p is an important regulator, which has a strong effect on the G1/S phase of cell cycle transition [16]. MiR-17 has been found to target certain genes in some cancers, such as bladder cancer and oral squamous cell

been found to be associated with colorectal cancer [13–15], there is still a lack of relevant studies exploring its regulatory mechanisms in colon cancer. As for miR-17-5, it is the first time to discover it was negatively related to tumor progression.

�en, using the target prediction method in the GCBI online tool, 1738 genes were selected as target genes for these DE-miRNAs. �e target genes predicted by GO analysis were enriched in “transcription, DNA-dependent”, “transcriptional regulation, DNA-dependent”, “signal transduction” and “positive regulation of transcription from RNA polymerase II promoter”. Interestingly, we noticed the opposite GOs

Table 2: �e top 10 dysregulated GOs.

Rank GO ID GO name Count FDR1 GO:0006351 Transcription, DNA-dependent 278 1.54E−602 GO:0006355 Regulation of transcription, DNA-dependent 169 3.34E−273 GO:0007165 Signal transduction 165 1.81E−374 GO:0045944 Positive regulation of transcription from RNA polymerase II promoter 154 5.61E−525 GO:0006915 Apoptotic process 111 4.75E−276 GO:0045893 Positive regulation of transcription, DNA-dependent 103 1.14E−327 GO:0000122 Negative regulation of transcription from RNA polymerase II promoter 103 2.41E−318 GO:0007399 Nervous system development 75 1.70E−309 GO:0007411 Axon guidance 75 4.27E−2710 GO:0006468 Protein phosphorylation 74 1.61E−24

Figure 5: miRNAs-GO-network. �e miRNAs-GO-network was generated according to the relationship of significant biological functions and miRNAs. �e yellow and blue circles represent GOs, red square nodes represent upregulated miRNAs, and blue square nodes represent downregulated miRNAs. �e size of the circle or square node represents the degree value. A higher degree of GO/miRNAs indicates that it plays a more important role in the signaling network.

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carcinoma [17, 18]. miR-182-5p is a member of the miR-183/96/182 cluster. Previous studies have identified its important role in breast cancer, glioma, prostate cancer, prostate cancer and renal cell carcinoma [19–23]. Generally, miR-182-5p regulates the apoptosis of tumor cells by targeting certain special genes, such as FOXO1, MTSS1, HMGA2, CASP9, and FOXO3 [24, 25], and these target genes were also predicted by our experiment. miR-200a-3p has been found to play important roles in the epithelial to the mesenchymal transition process in the development of cancer [26, 27].

Table 3: �e top ten dysregulated pathways of the target genes of DE-miRNAs.

Rank Pathway ID Pathway name Count FDR

1 04010 MAPK signaling pathway 69 2.00E−28

2 05200 Pathways in cancer 64 8.44E−19

3 04151 PI3K-Akt signaling pathway 61 1.17E−15

4 05205 Proteoglycans in cancer 47 5.80E−15

5 05166 HTLV-I infection 47 2.77E−126 04144 Endocytosis 45 2.58E−15

7 05202Transcriptional misregulation in

cancer38 1.97E−12

8 04722 Neurotrophin signaling pathway 33 3.08E−14

9 04360 Axon guidance 31 1.38E−11

10 04912 GnRH signaling pathway 24 6.40E−10

Table 4:  Hub miRNAs in miRNAs-gene-networks and miR-NAs-GO-networks.

Rank miRNAs FeaturemiRNA-gene-

networks degree

miRNA-GO- networks

degree1 miR-17-5p down 277 4492 miR-182-5p down 177 4053 miR-200a-3p down 126 342

Figure 6: In GSE83924, miRNAs expression levels of miR-17-5p (a), miR-182-5p (b), and miR-200a-3p (c) between normal colon and tumor samples. In GSE48267, miRNAs expression levels of miR-17-5p (d), miR-182-5p (e), and miR-200a-3p (f) between normal colon and tumor samples. Independent sample T test was used to evaluate the statistical significance of differences.

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Authors’ Contributions

Dong-Hu Yu and Wei Li are co-first authors. X.R., and S.L. conceived and designed the study, D.Y., W.L., and X.L. per-formed the analysis procedures, D.Y., W.L., J.H., C.Z., and X.L. analyzed the results, X.L., X.R., and S.L. contributed analysis tools, D.Y., and W.L. contributed to the writing of the manu-script. All authors reviewed the manuscript.

Funding

�is work was supported by the 351 Talent Project of Wuhan University (Luojia Young Scholars: SL) and Young & Middle-aged Medical Key Talents Training Project of Wuhan.

Supplementary Materials

Supplementary 1. Table S1: the details of GSE98406, GSE83924, GSE48267 and GSE35834. Supplementary 2. Table S2: putative target genes of DE-miRNAs. Supplementary 3. Table S3: clinicopathological correlation analysis for GSE35834.

miR-200a-3p plays a role like a tumor suppressor and its target genes are enriched in signal transmission and cell apoptosis control. miR-200a-3p is rarely used as a research focus and related regulatory mechanisms remain to be clarified.

In summary, we identified three hub miRNAs (hsa-miR-17-5p, hsa-miR-182-5p, and hsa-miR-200a-3p), which were closely related to the development of colon cancer. �e hub miRNAs we identified might provide references for the func-tional study of downstream proteins in other study and some clinical targeted treatments in the future. However, due to the small sample size of this study, these results still have certain limitations. Further, in vivo and in vitro studies are needed to understand the exact molecular mechanisms that influence the development of colon cancer.

Data Availability

�e data supporting the results reported in this article can be available by contacting the corresponding author.

Conflicts of Interest

�e authors declare that they have no conflicts of interest.

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Figure 7: ROC curve of miR-17-5p, miR-182-5p and miR-200a-3p in the datasets of GSE83924 and GSE48267.

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